JP2694840B2 - Method for expressing polypeptide by microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to heterologous genes encoding the expression of mammalian hormones such as somatostatin and other polypeptides, including plasmids suitable for transformation of bacterial hosts.
A method for producing a polypeptide using a recombinant microbial cloning vehicle containing ologous DNA (this plasmid contains a homologous regulatory region for the host in the untransformed state). And in the decoding phase, Heterologous D
It contains the structural gene for NA).

The invention also relates to a protein consisting of (a) a polypeptide hapten and an additional protein of sufficient size to confer immunogenicity to the expressed product, which is an antibody to the hapten for analysis. Or produce
(Which may be used to produce vaccines), and (b) a protein consisting of the desired polypeptide product and an additional protein capable of cleaving the desired product,
A method for producing a protein using a cloning vehicle encoding microbial expression.

The invention further provides a method for preparing a synthetic structural gene encoding the expression of mammalian polypeptides in a microbial cloning system.

Genetic information is encoded in a double-stranded deoxyribonucleic acid according to the sequence order in which the coding strand of DNA represents the characteristic base of the repeating nucleotide component. The "expression" of the encoded information to produce a polypeptide consists of a two-step process. That is, the RNA polymerase moves along the coding chain according to the instruction of the “regulatory region”, which is a regulatory region in a gene, and messenger RNA
(Ribonucleic acid) is produced. This process is called transcription. In the subsequent "translation" process, cellular ribosomes bound to transfer RNA convert messenger RNA (mRNA) messages into polypeptides. The information transcribed from mRNA by DNA includes signals for initiation and termination of ribosome translation as well as signals for identification and sequence of amino acids constituting the polypeptide. A DNA coding chain consists of a long chain of nucleotide triads called "codons" because the characteristic bases of nucleotides code specific information bits. For example, three nucleotides, which are read as ATG (adenine-thymine-guanine), are decoded by mRMA as a "translation start" signal, and stop codons TAG, TAA, and TGA are decoded by "translation stop". . Between this start codon and stop codon is a so-called structural gene, which determines the final translated amino acid sequence. This decision is made according to the already well understood "genetic code". This is described, for example, in J. et al., Which describes codons for various amino acids. D. Watson's Molecular
Biology of the Gene (WA Benjamin Inc.,
N. Y., 3rd ed. 1976). This genetic code is
It is "degenerate" in the sense that different codons produce the same amino acid, but is accurate in the sense that there is and is one or more codons for each amino acid. Therefore, for example, TTT, TT
All codons such as C, TTA and TTG specify serine when read as such and no other amino acids. During transfer, the proper reading phase, ie the open reading frame, must be retained. This means that, for example, in the case of the sequence GCTGGTTGTAAG ... When the transcript by RNA polymerase begins to read the start of the codon (underlined part) from different bases (following formula) It can be understood.・ ・ ・ ・ ・ ・GCT GGT TGT AAG・ ・ ・ → Ala-Gly-Cys-Lys ・ ・ ・ ・ ・ ・ G CTG GTT GTA AG ・ ・ ・ → Leu-Val-Val ・ ・ ・ ・ ・ ・ GC TGG TTG TAAG・ ・ ・ → Trp-Leu- (s
top)

Thus, the finally produced polypeptide is significantly affected by the spatial relationship between the structural gene and the regulatory region.

In order to better understand the process of genetic expression, the components of the gene are defined below. Structural genes for operon polypeptide expression and regulatory regions for controlling this expression, or "regulatory regions"
A gene consisting of. A gene within the control region to which RNA polymerase must bind in order to initiate promoter transcription. A gene to which an operator repressor protein can bind, which prevents RNA polymerase from binding to a promoter adjacent to this operator. Inducer A substance that inactivates the repressor protein, which releases the operator, allowing RNA polymerase to bind to the promoter and initiate transcription. Catabolic Metabolite Activating Protein (CAP) Binding Site CA
Cyclic adenosine monophosphate (c-AMP) mediated by P
A gene that binds to and is also normally required for initiation of transcription. This CAP binding site is not needed in special cases. For example, a promoter mutation in the lactose operon of phage λplac UV5 (abbreviated as lac-operon) eliminates the need for c-AMP and CAP for expression (J. Beckwith et al.
J. Mol. Biol. 69 , ISS-160 (1972)). Promoter operator system Here, CAP
It refers to a regulatory region of an operon that can be genetically engineered, whether or not it contains a binding site, and whether or not it has the ability to encode expression of a repressor protein.

Furthermore, the terms necessary for the discussion on the recombinant DNA described below are also defined below. A non-chromosomal double-stranded D containing a replicable intact replicon when placed in the unicellular organism "microbe" by a process called cloning vehicle transformation.
NA. The organism transformed in this way is transformed into a transformant (tra).
nsformant). Plasmid In the present invention, a cloning vehicle derived from a virus or a bacterium, and in the case of a bacterium, it is called a bacterium (bacterium) plasmid. A characteristic base sequence of complementary single-stranded DNA has the nature that allows the double-stranded DNA is formed by hydrogen bonding between complementary bases on the respective strands. Adenine (A) is complementary to thymine (T), and guanine (G) is complementary to cytosine (C).

Recent advances in biochemistry have made it possible, for example, to assemble (construct) "recombinant" cloning vehicles in which the plasmid contains exogenous DNA. In special cases, this recombinant may contain heterologous DNA. Heterologous DNA means DNA encoding a polypeptide not normally produced by an organism transformed with a recombinant vehicle. For example, the plasmid is cleaved to obtain linear (linear) DNA with ligable ends. This is ligated with an exogenous gene having a ligable end to obtain a replicon as it is and one biologically functional part having desired phenotypic characteristics. This recombinant portion is inserted into a microorganism and transformed, and this transformant is isolated and cloned to obtain a large population capable of expressing new genetic information. Many literatures describe methods and means for forming a recombinant cloning vehicle and transforming a microorganism with it.

The documents and the like referred to in this specification are listed below for reference. HL Heynecker et al, Nature 2
63 , 748-752 (1976); Cohen et al, Pro.
c. Nat. Acad. Sci. USA 69 , 2110 (197)
2); ibid. 70 1293 (1973); ibid. 70 , 3240 (1)
973); ibid. 71 1030 (1974); Morrow et a
l, Proc. Nat. Acad. Sci. USA 71 , 1743,
(1974); Novick, Bacteriological Rev. 33 ,
210 (1969); Hershfield et al, Proc. Soc.
Nat'l. Acad. Sci. USA 71 , 3455 (197)
4) and Jackon et al, ibid, 69 , 2904 (197).
2).

For the recombination of DNA, various methods can be used to repair the adjacent ends of the separated DNA fragments by some method to facilitate the ligation. This ligation refers to the formation of a phosphodiester bond between adjacent nucleotides, most commonly by the enzyme T ₄ DNA ligase (synthetic enzyme). In this way, blunt ends can also be directly attached. Alternatively, if they have fragments containing complementary single strands at their flanking ends, the conjugation is advantageously effected by hydrogen bonding locating the respective ends for subsequent conjugation. Sticky end
Such single strands, called (or complementary ends), may be formed by adding nucleotides to the blunt end using a terminal transferase, optionally with one strand of the blunt end of the λ-exonuclease. It may be formed by simply destroying with an enzyme such as. Rather, most commonly, restriction endonucleases that cleave phosphodiester bonds within and around specific nucleotide sequences of about 4 to 6 base pairs in length.
ucleases). Many restriction endonucleases and their recognition sites are known, the so-called E
The co RI endonuclease is the most widely used. Double-stranded DNA is converted to a palindromic "regression point" (palindr) for rotation.
Restriction endonucleases that are cleaved by omes) leave sticky ends. Thus, plasmids or other cloning vehicles can be cleaved leaving an end each consisting of half a restriction endonuclease recognition site. Cleavage products of exogenous DNA obtained by the same restriction endonucleases will have ends complementary to those at their plasmid ends. Alternatively, as described below, synthetic DNA having sticky ends
It can be inserted into this cleaved vehicle. Until the foreign DNA is inserted, this end may be digested with alkaline phosphatase to prevent the sticky ends of the vehicle from recombining, thus ending the contamination of the foreign fragment. Molecular selection is performed. Insertion of a fragment with the proper orientation with respect to the other orientation of the vehicle
It can be enhanced if it replaces the vehicle DNA removed by two different restriction endonucleases, each of which itself consists of ends that make up half of the recognition sequence of the different endonuclease.

Recently, extensive research has been conducted on recombinant DNA, but few results that can be applied immediately and practically have been obtained. This was encoded by "synthetic DNA", whether assembled one by one by conventional means or obtained by reverse transcription from isolated m-RNA (complementary, cDNA). This is especially true in that attempts to express polypeptides and the like have failed.

Described herein are what appear to be examples of the first expression of a functional polypeptide product from a synthetic gene, and related new facts that promise widespread application. This product is somatostatin (Guillemin) which is a growth hormone secretion inhibitor.
U.S.P. 3,904,594), insulin and glucagon, the results of which suggest their application in the treatment of apical pain, acromegaly, acute pancreatitis and insulin-dependent diabetes mellitus (R. Guillemin et al. al, Annual Rev. Me
d. 27 379 (1976)).

As will be apparent from the accompanying drawings and the description detailed below, the somatostatin model described herein demonstrates that the novel facts described herein can be applied in many beneficial ways.

DESCRIPTION OF THE FIGURES The accompanying drawings illustrate one mode of use of the present invention, namely the expression of somatostatin hormone and human insulin by bacterial transformants containing recombinant plasmids. It is an example.

FIG . 1 is a schematic diagram of the method. Chemical DNA
The synthetically prepared gene for somatostatin was cloned into E. coli β on plasmid pBR322.
-Fused with the galactosidase gene. After transformation into E. coli, this recombinant plasmid is capable of selectively cleaving methionine residues with cyanogen bromide in vitro, thus producing an active mammalian polypeptide hormone. Directs the synthesis of protein precursors. A, T, C, and G represent the unique bases of deoxyribonucleotides in the coding chain of the somatostatin gene (A represents adenine, T represents thymine, C represents cytosine, and G represents guanine, respectively).

FIG . 2 Schematic structure of structural gene. The coding strand (ie the upper strand) consists of codons for the amino acid sequence of somatostatin (shown in the figure).

FIG . 3 is a schematic representation of a preferred method for preparing the nucleotide trimer used to assemble the structural gene. In FIG. 3, the normal notation was used to depict the nucleotides, 5'OH on the left and 3'OH on the right as shown below.

Embedded image

FIG . 4 pBR322 as parent plasmid
Is used to express a somatostatin (represented by SOM in the figure) -containing protein.
Flowchart for preparing M11-3). In Fig. 4, the approximate molecular weight of each plasmid is expressed in daltons (d).
Indicated by Ap ^r and T c ^r represent the genes for ampicillin and tetracycline resistance respectively,
Tc ^s represents tetracycline sensitivity derived from excision of a part of the Tc ^r gene. The relative positions of various restriction endonucleases specific for the cleavage site on the plasmid are shown in the figure (eg Eco RI, BamH
I).

Figures 5A and B show the nucleotide sequences of the major sites of the two plasmids . The direction of messenger RNA (mRNA) transcription is also indicated. This transcription always starts from the 5'end of the coding chain. Restriction endonuclease substrate sites are also shown. The sequences depicted contain both the regulatory elements of the lac (lactose) -operon and the codons for expressing the amino acid sequence of somatostatin (italics), respectively. The amino acid sequence numbers for β-galactosidase (hereinafter β-gal) are shown in brackets.

FIGS. 6-8, which are described in detail in the experimental section below, show the results of comparative experiments of radioimmunoassays, which show the products expressed by the recombinant plasmids. It shows somatostatin activity.

FIG . 9 The coding chain is human insulin A
Schematic structure of a synthetic gene consisting of codons for the chain and B chain amino acid sequences.

FIG . 10 is a flow chart for constructing a recombinant plasmid capable of expressing the B chain of human insulin.

Detailed Description 1. Preparation of Gene Encoding a Heterologous Polypeptide DNA encoding a polypeptide whose amino acid sequence is known can be prepared by selecting codons according to the "genetic code". To facilitate purification and the like, oligodeoxyribonucleotide fragments consisting of, for example, about 11 to about 16 nucleotides are prepared separately and then assembled into the desired sequence. Thus, conveniently sized first and second sets of oligodeoxyribonucleotide fragments are prepared.
This first set, when ligated with the appropriate sequences, provides the DNA coding strand for polypeptide expression (see, eg, fragments A, B, C and D in Figure 2). The second set also results in a strand complementary to the coding strand when attached in the same order (see, eg, fragments E, F, G, and H of Figure 2). Fragments of each strand are conveniently overlapped by complementarity, so that the cohesive ends (complementary ends) of the fragment blocks are hydrogen-bonded to automatically assemble. Subsequent to assembly, the structural gene is completed by ligation in a usual manner.

In selecting a codon for a given amino acid sequence, a considerable degree of freedom is allowed due to the degeneracy of the genetic code. However, for the purposes of the present invention, the choice of codon was conveniently determined according to the following three conditions. First, except for fragments that are adjacent to each other in the desired gene, codons and fragments were selected so as to avoid inappropriate complementarity between fragments, and fragment assembly was performed. The second is to avoid sequences that are rich in AT base pairs (eg, about 5 or more) so that transcription does not terminate prematurely, especially if GC base pair rich sequences are present first. Is. Third, at least most of the selected codons
Preferred for expression of microbial genomes (eg W. Fiers, et al, Nature 260 500 (197)
6)). In the present invention, codons suitable for expressing a microbial genome are defined below.

[0025]

[Table 1] Designation of preferred codons 1st character (5 'end) 2nd character ( read across ten characters) 3rd character (3' end) (read below) T C A G (read below) phe − − Cys T T phe ser tyr − C leu − end stop A − ser end trp G leu pro his arg T C leu pro his arg C leu pro gln − A − pro gln − G ile thr asn − T A ile thr asn ser C − − − − A met (start) thr lys − G val ala asp gly T G val − asp − C val − glu − A val ala glu − G

The relationship between the most preferred amino acid (codon) and structural gene in the case of somatostatin is as follows. gly (GGT), cys (TGT), lys (AAG), trp (T
GG), ala (GCT, GCG), asn (AAT, AAC), p
he (TTC, TTT), thr (ACT, ACG), ser (TC
C, TCG)

When the structural gene for the desired polypeptide is directly inserted into a cloning vehicle for expression, this gene is preceded by a start codon (eg, ATG) immediately followed by one or more terminations, That is, put a stop codon (see FIG. 2).

As a structural gene suitable for expression as it is, the procedure of L.C. (Li, C.)
Of National Academy Sciences (Proc.N
atl.Acad.Sci.) 73 volumes, 1476-1479, 197
DN, which can be synthesized in the same manner as the present invention, based on the mature amino acid sequence of human growth hormone disclosed in 2006.
A, or Proceedings of National Academy of Sciences, Higuchi et al., USA, 73, 3
146-3150, disclosed in 1976, is a gene encoding rabbit beta-globulin, and the like. However, as described below, the amino acid sequence of a particular polypeptide may be expressed with additional proteins preceding and / or following.

In the present specification, a structural gene of a desired polypeptide with start and stop codons is referred to as "DNA encoding the amino acid sequence of heterologous polypeptide", and an expression product of the DNA alone is referred to. The "desired heterologous polypeptide itself" and the expression product of the DNA to which the homologous gene or a fragment thereof is added are referred to as "precursor polypeptide". In this precursor polypeptide, if it is necessary to cleave an additional protein for the purpose of using the polypeptide, an appropriate cleavage site should be provided at the junction of the adjacent polypeptide and the additional protein codon. Encrypt. Thus, for example, in Figure 1, the expression product is a protein precursor consisting of both somatostatin and most of the β-galactosidase polypeptide. In this case, it is not necessary to encrypt the ATG to start the translation. This is because the decoding of additional β-gal protein by the ribosome is read through to the somatostatin structural gene. However, the insertion of the ATG signal, which provides the code that prepares the amino acid methionine, which is specifically cleaved by cyanogen bromide, allows for easy conversion of the protein precursor into the desired polypeptide.

FIG. 2 also shows a heterologous DNA used for recombination.
Another feature that is preferred for is that, advantageously, there is a sticky end consisting of one half of the double-stranded restriction endonuclease recognition site. For the reasons described above, it is preferable to design both ends so that they form different recognition sites upon recombination.

The new facts described here are preferably exemplified by using the somatostatin model. However, if the amino acid sequence is actually known, the heterologous DNA code for it is required. Make a change,
It should be readily understood that it can be used. For example, the techniques described above and below describe, by mutatis mutandis, poly (amino) acids such as polyleucine and polyalanine, enzymes, serum proteins, β-endorphins that alter the threshold of pain.
It can be used for the production of analgesic polypeptides such as (β-endorphins). Most preferably, the polypeptides thus produced will be mammalian hormones or intermediates thereof. Such hormones include, for example, somatostatin, human insulin, human and bovine growth hormone, stromal cell stimulating hormone, AC.
TH, pancreatic polypeptides and the like are included. Examples of the intermediate include human preproinsulin, human proinsulin, human insulin A chain and B chain, and the like. As the heterologous DNA, DN prepared in vitro is used.
In addition to A, cDNA obtained by reverse transcription from mRNA
A may also be included (eg Ullrich et al. Science
196 1313 (1977)).

[0032] 2. Encode the expression of protein precursors
In the process schematically depicted in Figure 1, the expression consists of a polypeptide (somatostatin) encoded by a unique heterologous structural gene and an additional protein (a protein of the β-galactosidase enzyme). A protein precursor consisting of both is produced. The desired polypeptide can then be separated from unwanted (excess) proteins by selective cleavage sites flanking the somatostatin amino acid sequence. The illustrated case is representative of the various methods that can be used with the techniques described herein.

In most cases, the cleavage is performed outside the replication environment of the plasmid or other vehicle, eg after harvest of the microbial culture. In this way, temporary conjugation of the small polypeptide to unwanted proteins can prevent the small polypeptide from being degraded by, for example, a native enzyme in vivo. At the same time, this additional protein normally abolishes the biological activity of this desired polypeptide until it is cleaved extracellularly, which has the effect of increasing biological safety during the procedure. Of course,
In special cases it may be desirable to cleave it intracellularly. For example, DNA encoding the enzyme that converts the insulin precursor into its active form can be added to the cloning vehicle by serially treating it with the DNA encoding the expression of the precursor.

It is preferable that the specific polypeptide of interest does not contain a cleavage site in the molecule corresponding to the cleavage site used for eliminating an unwanted protein. It will be appreciated that, if not satisfied, the competition reaction will give the desired product, albeit in low yield. If the desired product does not contain methionine, it has been found to be very effective to cleave with methionine adjacent to the desired sequence with cyanogen bromide. Similarly, if the product is free of arginine and lysine, trypsin or chymotrypsin, for example, can be used to enzymatically cleave cleavage sites such as arg-arg, lys-lys, which flank the desired sequence. If the cleavage yields the desired product, for example with unwanted arginine attached, this arginine can be removed by digestion with carboxypeptidase. When trypsin is used to cleave arg-arg, the lysine site in the polypeptide of interest can be protected beforehand with maleic anhydride or citraconic anhydride. It will be readily apparent to those skilled in the art in light of this specification that the cleavage techniques described herein by way of example are merely representative of many variations.

The cleavable protein may be expressed adjacent to either the C-terminus or the N-terminus of a particular polypeptide, or alternatively, of an included sequence that distinguishes proinsulin from insulin. As in the case, it may be expressed within the polypeptide itself. In addition, the vehicle used may consist of a repetitive sequence of the desired polypeptide, and may be encoded so as to express a protein in which each peptide is separated by a specific cleavage site. However, it is most preferred that the codon for the unwanted protein is translated before the structural gene of the desired product, as is the case illustrated in the figure. In each case, care must be taken to maintain the proper open reading frame (reading phase) for the control area.

[0036] 3. Expression of Immunogenic Substances The ability to express both specific polypeptides and unwanted proteins can be a powerful tool for preparing immunogenic substances. The polypeptide "hapten" (adhesive, that is, a substance that has a determinant that specifically binds to an antibody, etc., but is usually too small to show an immune response) is sufficient to confer immunogenic potential. It can be expressed as a conjugate with additional proteins of size. In fact, as an example, the β-gal-somatostatin conjugates prepared here are immunogenic in size and can be expected to produce antibodies that bind the somatostatin hapten. 10
Proteins consisting of 0 or more amino acids, most commonly 200 or more amino acids, are immunogenic.

The conjugate prepared by the method described above produces antibodies which are useful in the radioimmmune assay or other assays of haptens, or alternatively in the production of vaccines. The latter application example will be described below. Cyanogen bromide or other cleaved substances of the viral coat protein produce oligopeptides that bind to antibodies raised against the protein itself. If the amino acid sequence of such oligopeptide hapten is known, the heterologous DNA therefor can be expressed as a conjugate with an additional protein imparting immunogenic ability. The use of such a conjugate as a vaccine is expected to reduce the side effects that occur when the outer coat protein itself is used for immunization.

[0038] 4. Regulatory Elements FIG. 1 represents the process by which a transformed organism expresses a polypeptide product from inserted heterologous DNA under the control of a regulatory region which is homologous to the organism in its untransformed state. That is, the chromosomal DNA of lactose-dependent Escherichia coli is, inter alia, the lactose operon that performs lactose digestion by producing the enzyme β-galactosidase, that is, lac.
-Contains an operon. In this illustrated example, this la
The c-regulatory element is derived from the bacteriophage λplac5 that infects E. coli. On the other hand, the lac-operon of this phage was induced by transduction from the same bacterial species and is therefore "homology". The homologous control region preferably used in the described method is also the plasmid D derived from that organism.
It may be derived from NA.

Since this lac-promoter-operator system is simple and efficient, it is desirable to use it in the above system. This system also has its function (ability)
Is also desirable in that it is induced by IPTG (isopropylthio-β-D-galactosidase).
Of course, other operons or parts thereof, such as lambda
(lambda) promoter - operator, Arabic North operon [phi 80 Dhara (phi 80 d ara)] or colicin (colicine) E1, galactose can also be used, such as an operon alkaline phosphatase or tryptophan. The promoter-operator from the tryptophan operon (trp operon) is inducible (inductant).
ion) (with indole acrylic acid) and harvest 10
It is expected to suppress by 0%.

5. The details of the procedure schematically shown in FIG. 4 for the overall plasmid assembly will be described in the experimental section. Here is a brief description of the various techniques used to construct the preferred embodiment recombinant plasmids.

Two plasmids were used for cloning and expression of the synthetic somatostatin gene. Each plasmid has an Eco RI substrate site in a different region of the β-galactosidase structural gene region.
(See FIGS. 4 and 5). Eco of these plasmids
Insertion of a synthetic somatostatin DNA fragment at the RI site results in expression of the genetic information in that fragment under the control of the lac-operon regulatory element. Insertion of the somatostatin fragment into these plasmids resulted in a translation of 10 amino acids (pSOM1).
Or virtually all β-galactosidase subunit structures
One obtains a somatostatin polypeptide directed by either (pSOM1 1-3).

This plasmid assembly design begins with the well characterized cloning vehicle, plasmid pBR322. To introduce the lac-element into the plasmid, the lac-promoter, CA
H with a P (cyclic AMP receptor protein) binding site, an operator, a ribosome binding site and the first 7 amino acid codons of the β-galactosidase structural gene
This was done by inserting the aeIII restriction endonuclease fragment (203 nucleotides). This HaeIII
The fragment was obtained from λplac5 DNA. The end is T
₄ EcoRI-cleaved pBR322 plasmid repaired with ₄ DNA polymerase and deoxyribonucleotide triphosphate was blunt-end ligated with this HaeIII fragment to generate an EcoRI end at the point of insertion. These HaeII
Coupling of the I and repaired Eco RI ends creates an Eco RI restriction site at each end (FIGS. 4 and 5).
reference). E. coli with this DNA Transformants of E. coli RRI were selected by resistance to tetracycline (Tc) and ampicillin (Ap) on 5-bromo-4-chloro-indolylgalactoside (X-gal) medium. On this indicator medium, it binds to the repressor (titrati
ng) lac-increased number of operators
-A colony capable of constitutively synthesizing galactosidase is identified by its blue color. HaeII
The I fragment has two possible orientations, which are distinguished by the asymmetric presence of the Hha restriction site in the fragment. Plus mid pBH10
To further modify the lac-operator distal Eco
The RI endonuclease site was removed (pBH20).

The 5'end [ ³² P] -γ-ATP of eight chemically synthesized oligodeoxyribonucleotides (FIG. 2)
Labeled with polynucleotide kinase using, T ₄ D
It is bound by NA ligase. Hydrogen bonding between overlapping fragments causes the somatostatin gene to automatically assemble and ultimately polymerize by sticky restriction site ends into larger molecules. This combined product is Eco
Treated with RI and BamHI restriction endonucleases,
Generate the somatostatin gene as shown in FIG.

This synthetic somatostatin gene fragment with EcoRI and BamHI ends is ligated with pBH20 plasmid which had been previously treated with EcoRI and BamHI restriction endonucleases and alkaline phosphatase. This treatment with alkaline phosphatase allows for molecular selection for the plasmid carrying the inserted fragment. The resulting ampicillin-resistant transformants with this ligated DNA were screened for tetracycline sensitivity and, in some cases, for the insertion of the appropriate size EcoRI-BamHI fragment.

EcoRI-from two clones (branching system)
Both strands of the BamHI fragment were labeled with BamHI and E
It was analyzed by nucleotide sequence analysis starting from the coRI site. This sequence analysis was extended to lac-regulatory elements. As a result, the lac-fragment sequence remained intact. In one case, in the case of pSOM1, the nucleotide sequences of both chains were determined separately, but each has the sequence shown in FIG. 5A.

The EcoRI-Pst fragment of the pSOM1 plasmid containing the lac-regulatory element was removed to give pBR3.
Plasmid pSOM11 was prepared by substituting 22 EcoRI-Pst fragments. The EcoRI fragment of λplac5, which contains the lac-operon regulatory region and most of the β-galactosidase structural gene, was inserted into the EcoRI site of pSOM11. EcoRI lac- of λplac5
There are two possible directions for the fragment. One of these two orientations maintains a proper open reading frame up to the somatostatin gene, while the other does not. Separately isolated clones were analyzed for somatostatin activity and clones containing genes with the proper orientation were identified. It is pSOM11-
The clone was certified as 3.

6 Potential candidates for transformation of microorganisms include various unicellular microorganisms such as bacteria, fungi and algae. That is, it is a unicellular organism that can grow by culture or fermentation. Bacteria are often the most manageable organisms. Bacteria that are easily transformed include Enterobacteriaceae, such as Escherichia coli.
chia Coli strain and Salmonella strain, those of the Bacillus family (Bacillaceae), for example Bacillus subtilis (Bacill)
us subtilis), Streptococcus pneumoniae (Pneumococcus)
(Streptococcus) and Haemophilu
s influenzae).

The microorganisms selected in the somatostatin experiments described below were genotyped as Pro ^- Leu ^- Thi ^- RB.
^- MB recA ⁺ Str ^r Lacy ^- of E. coli strain RRI (E.
Coli. strain RRI). E. FIG. Coli RRI
E. coli as a high frequency recombinant donor (Hfrdonor). Coli
By crossing with the K12 strain KL16, E. Col
i HB101 (H.W. Boyer et al, J. Mol. Bio
l. (1969) 41 459-472). Regarding this, J. H. Milley, Experiments in Mole
cular Genetics (Cold Spring Harbor, New York,
1972).

E. Coli RRI and E. Coli. RR
I (pBR322) Both cultures are American Type Cu
deposited with the Iture Collection (ATCC). These strains are ATCC numbers 31343 and 3134, respectively.
It is available under the number 4. Somatostatin-producing bacteria,
E. FIG. Coli. RRI (pSOM11-3) was also deposited (ATCC number; 31447).

For human insulin, A and B
Chain gene, E.Coli K12 strain 294EndA (endonuclease ^{A), thi -, hsr -} , hsmK + [ATCC number;
31446]. This microorganism was used for expression of A chain (E. Coli K12 strain 294
[PIA1] [ATCC number; 31448]. The B chain of human insulin was first derived from a derivative of HB101, namely E. coli.
K12 strain D1210 lac ⁺ expressed by (iQo ⁺ z ^t y ⁺⁾ in. This B gene containing organism E. Coli K12 D121
0 (pIB1) was also deposited (ATCC number; 314
49). Alternatively, the B gene may be inserted into and expressed from the first mentioned organism, ie strain 294.

Further, these E. Coli strains have been deposited at the Institute of Microbial Engineering, Institute of Industrial Science and Technology, which has an address at 1-3, Higashi 1-Ta, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture. The accession number of each strain is as follows. Strain deposit number E. Coli RRI ...... 4709 E. Coli RRI (pBR322) ...... 4711 E. Coli K-12 294 ..... ........ 4710 E.Coli RRI (pSOM11-3) .... 4712 E.Coli K-12 294 (pIA1) ..... 4714 E.Coli K-12 D1210 (pIB1). 4713

Experiment I. Somatostatin 1. Construction of Somatostatin Gene Fragment Eight oligodeoxyribonucleotides, named A to H, shown in FIG.
(K. Itakura) et al., The Journal of the American Chemical Society (J. Am. Chem. Soc.)
It was first constructed by the modified triester method of Volume 97, page 7327 (1975). However, in the case of fragments C, E and H, one had to resort to an improved technique consisting of first producing a fully protected trimer as the building block for assembling longer oligodeoxyribonucleotides. In FIG. 3, which outlines this improved technique, B is thymine, N-benzoylated adenine, N-benzoylated cytosine or N-isobutyryl guanine. In summary, as shown in FIG. 3, a strong binder, namely 2,4,6-triisopropylbenzenesulfonyltetrazolid (TPSTe, 4 mmol; 2)
The coupling reaction of excess I (2 mmol) with II (1 mmol) in the presence of was almost complete in 60 minutes.
After removal of the 5'- protecting group with 2% benzene sulfonic acid solution, the 5'-hydroxyl dimer V during CHCl ₃ NaH
It could be easily separated from the excess of 3'-phosphodiester monomer IV by solvent extraction with CO ₃ solution. The fully protected trimer block is then
5'-hydroxyl dimer V, I (2 mmol) and TPST
prepared from e (4 mmol) and isolated by chromatography on silica gel (B. Hund (B.
T. Hunt) et al., Chemistry and Industry
(Chem. And Ind.) 1967, 1868 (1967)
Year)). The trimer yields produced by this improved technique are shown in Table 2.

[0053]

Yield of fully protected trimer Sequence yield Sequence yield TTT 81% ATG 69% TTT 75% GCC 61% GGA 41% CCA 72% AGA 49% CAA 72% ATC 71% TTA 71% CCT 61% CAT 52% ACA 63% CCC 73% ACC 65% AAC 59% CGT 51% GAT 60%

After removing all protecting groups, eight oligodeoxyribonucleotides were added to the permaphase (Permap).
hase) purified by high pressure liquid chromatography using AAX [RA Henry et al.
J. Chrom. Sci. Volume II, p. 358 (1973)]. The purity of each oligomer was determined by labeling the oligomer with [γ- ³² P] -ATP in the presence of polynucleotide kinase, followed by homochromatography using thin layer DEAE-cellulose and electrophoresis in a 20% acrylamide slab. Checked. One major labeled product was obtained from each DNA fragment.

2. Somatostatin DNA binding (ligatio
n) and 5'OH ends acrylamide gel analysis chemically synthesized fragments A to H, were separately pressurized phosphorylated with T ₄ polynucleotide kinase. [ ³² P] -γ-ATP was used in the phosphorylation reaction so that the reaction product could be traced by autoradiography.
Unlabeled AT without relying on autoradiography
It should be easily understood that P can also be used, but immediately before the kinase reaction, [γ- ³² P] ATP 2
5 μCi (about 1500 Ci / mmol) [Maxim]
And Gilbert, Proceedings of the National Academy of Sciences of the United States of America (Proc.Nat.Acad.Sci.U.S.A.) Volume 74, 15
07 pages (1977)] to 0.5 ml of Eppendorf (Eppe
ndorf) tube was evaporated to dryness. 5 μg fragment
2 units of T ₄ DNA kinase (hydroxyl apatite fraction, 2500 units / ml) and a total amount of 15
0 μl pH 7.6 Tris-hydrochloric acid 70 mmol, MgCl ₂
Incubated in 10 mM, 5 mM dithiothreat for 20 minutes at 37 ° C. To ensure that the fragment used for ligation was phosphorylated as much as possible, 70 mM Tris-HCl pH 7.6, MgCl ₂ 1
0 mmol, dithiothreat 5 mmol, ATP 0.
10 μl of a mixture consisting of 5 mmol and 2 units of DNA kinase was added and the incubation was continued at 7 ° C. for 20 minutes. This fragment (250 μg / μl) was stored at −20 ° C. without further treatment. Fragments A, B, E and F that have undergone kinase reaction (each 1.25 μm
g) in a total volume of 50 μl, 20 mM Tris-HCl, pH 7.6, 10 mM MgCl _2, 10 mM dithiothreate, 0.5 mM ATP and T ₄ DNA ligase.
(Hydroxyl apatite fraction, 400 units / ml)
The binding was carried out at 4 ° C. for 16 hours. Fragments C, D, G and H were also ligated under similar conditions. A 2 μl sample was taken, electrophoresed on a 10% polyacrylamide gel and then analyzed by autoradiography [H.L. Heyneker et al., Nature.
e) Volume 263, p. 748 (1976)]. Unreacted DN
The A fragment appears as a fast-running substance, and the monomeric body of the bound fragment migrates with bromphenol blue dye (BPB). Bound fragments A, B, E and F and bound fragment C,
Some dimerization occurs due to the sticky ends of D, G and H. These dimers appear as the slowest electrophores and can be cleaved by the restriction endonucleases EcoRI and BamHI, respectively.

The two halves of the molecule (bound A + B +
E + F and bound C + D + G + H) at 4 ° C. for 16
The binding was performed for an additional time with an additional coupling step performed in a final volume of 150 μl. 1 μl was taken for analysis. T ₄ D
The reaction mixture was heated to 65 ° C. to inactivate the NA ligase.
Heated for 15 minutes. This heat treatment does not affect the migration pattern of the DNA mixture. Add sufficient amount of the restriction endonuclease BamHI to the reaction mixture and allow 30 minutes at 37 ° C. for somatostatin DNA multimers (multimeric form).
Cleaved. After adding 100 mM NaCl, the DNA was digested with EcoRI endonuclease. The restriction endonuclease digestion was terminated by extracting the DNA with phenol-chloroform. Unreacted somatostatin DNA fragment
And preparative using 10% polyacrylamide gel to separate and partially bound DNA fragments
Purification was carried out by (preparative) electrophoresis. The band containing the somatostatin DNA fragment is cut from the gel, the gel is sliced into small pieces and the DNA is eluted with elution buffer.
(Ammonium acetate 0.5 mol, MgCl ₂ 10 mmol,
The DNA was eluted by extraction with 0.1 mmol EDTA, SDS (0.1% sodium dodecyl sulfate) at 65 ° C. overnight. This DNA was precipitated by adding 2 volumes of ethanol, centrifuged, redissolved in 200 μl of 10 mM Tris-HCl having a pH of 7.6, and dialyzed against the same buffer to give a somatostatin DNA concentration of 4 μg / ml.
It became.

3. Construction of Recombinant Plasmid FIG. 4 shows an outline of the method for constructing a recombinant plasmid containing the somatostatin gene, which will be described in detail below.

A. Parental plasmid pBR322 The plasmid selected for experimental somatostatin cloning was pBR322, which is a small (molecular weight approximately 2.6 megadalton) plasmid with resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). is there. As shown in FIG. 4, the ampicillin resistance gene has a cleavage site by the restriction endonuclease PstI, the tetracycline resistance gene has a similar cleavage site by the restriction endonuclease BamHI, and the EcoRI site is Ap ^r. It is located between the Tc ^r. This plasmid pBR322 is a Ap ^r Tc ^r Col ^imm plasmid of 5.8 Mega Dalton pBR313
Derived from [R-L-Rodriguez (RLR
odriquez) et al., I N-Yu She L.
A Symposia on Molecular and Cellular Biology (ICN-UCLA Symposia on
Mloecular and Cellular Biology) Volume 5, 471-
77 (1976); Molecular Mechanisms in the Control of Gene Expressions.
eExpression) pp. 471-77, Academic Press, Incorporated (Academic Press, Inc. 1
(976)

B. Construction of plasmid pBH10 5 μg of plasmid pBR322 DNA was incubated at 37 ° C. for 30 minutes at a pH of 7.6, 100 mM Tris-HCl, NaCl 100.
Mmol, it was digested with the restriction endonuclease EcoRI10 units MgCl ₂ 6 mM in. The reaction is terminated by phenol-chloroform extraction, then DNA
The ethanol 2.5 volumes precipitated by addition, T ₄ D
NA polymerase buffer [pH 8.8 Tris-HCl 67 mmol, MgCl ₂ 6.7 mmol, (NH ₄ ) ₂ SO ₄ 16.6
Mmol, bovine serum albumin 167 μg / ml, dNT
P (deoxynucleoside triphosphate) 50 μmol each; A. Panet et al., Biochem., Vol. 12, 5045 (1973).
Reference] Resuspended in 50 μl. The reaction was initiated by adding 2 units of T ₄ DNA polymerase. After incubating for 30 minutes at 37 ° C, the DNA was extracted with phenol-chloroform to terminate the reaction, and then precipitated with ethanol. λplac ⁵ DNA [Shapiro et al., Nature 224, 768 (1969)] 3μ
g to a final volume of 20 μl pH 7.6 Tris-hydrochloric acid 6 mmol, MgCl ₂ 6 mmol, β-mercaptoethanol 6
In millimolar with the restriction enzyme HaeIII (3 units) 37
Digested at ° C for 1 hour. The reaction was terminated by heating at 65 ° C for 10 minutes. pBR322 treated DNA was mixed with HaeIII digested λplac ⁵ DNA in a final volume of 30 μl.
pH7.6 Tris-HCl 20 mM, MgCl ₂ 10 mM, dithio Torre It 10 mmol, in ATP0.5 mmol, 12 hours at 12 ° C., T ₄ DNA ligase (hydroxylapatite fraction; TA Panetto et al, supra) 1.
Blunt ends were ligated using 2 units. This combined DN
The A mixture was dialyzed against 10 mM Tris-HCl, pH 7.6, and E. It was used to transform E. coli strain RRI. The transformant was a 5-bromo-4-chloro-indolylgalactoside (X-gal) medium [JH Miller (JHM
iller), Experiments in Molecular Geneti
cs), Cold Spring Harbor (Cold Spling)
(Harbor), New York, 1972] Tetracycline and ampicillin resistant were selected on minimal medium plates containing 40 μg / ml. Colonies capable of synthesizing β-galactosidase were identified by their blue color. As a result of screening 45 independently isolated blue colonies, three of them were plasmid DNAs having two EcoRI sites separated by about 200 base pairs.
Was found to be contained. 203b.p. HaeIII lac-
An asymmetric HhaI fragment among regulatory fragments [W. Gilbert et al., Protein-Ligand Interactions (Protein-
Ligand Interactions), Etch Sand (H.sand)
And G. Blauer, co-edited, De Gruyter, Berlin, (1975) 19
The position of pages 3 to 210] can determine the orientation of the HaeIII fragment in these plasmids, here the orientation of the EcoRI fragment. It was found that the plasmid pBH10 contained the lac-regulatory region fragment in the desired orientation, that is, transcription proceeded to the tetracycline resistance gene under the control of this lac-regulatory region fragment.

C. Construction of plasmid pBH20 Next, plasmid pBH10 was modified to remove the EcoRI site at the end of the lac-operator. Tc ^r and lac which are separated by only about 40 bases pairs
-This was done by selective cleavage of this end site with EcoRI endonuclease, while partially protecting another EcoRI site located between the promoters with RNA polymerase. After binding RNA polymerase, this DNA (5 μg) was added to Eco in a final volume of 10 μl.
Digested with RI (1 unit) for 10 minutes at 37 ° C. 65
The reaction was terminated by heating at 0 ° C for 10 minutes. This Eco
The RI sticky ends were treated with S in 25 mmol sodium acetate, pH 300, 300 mmol NaCl, 1 mmol ZnCl _{2 at} pH 4.5.
Digested with 1 nuclease for 5 minutes at 25 ° C. The reaction mixture was treated with EDTA (10 mmol final) and pH8.
Tris-HCl (50 mmol final) was added to finish.
DNA was extracted with phenol-chloroform, precipitated with ethanol and resuspended in 100 μl of T ₄ DNA binding buffer. T ₄ DNA ligase (1 μl) was added and the mixture was incubated at 12 ° C. for 12 hours. Combined D
NA to E. Escherichia coli strain RRI was transformed with Ap ^r T
The ^cr transformants were selected on X-gal antibiotic medium. 1
Restriction enzyme analysis of DNA screened from 0 isolated blue colonies revealed that these clones had plasmid DNA with a single EcoRI site. Seven of these colonies retained the EcoRI site located between the lac and Tc ^r promoters. One of these plasmids, pB
The nucleotide sequence order of H20 from the EcoRI site to the lac-regulatory region was confirmed. This plasmid was then used to clone the somatostatin gene.

D. Construction of plasmid pSOM1 20 μg of plasmid pBH20 in a final volume of 50 μl
Restriction endonucleases EcoRI and BamHI in
Completely digested by. Add bacterial alkaline phosphatase [Worthington BAPE 0.
Incubation was continued for 10 minutes at 65 ° C.
The reaction was terminated by phenol-chloroform extraction, the DNA was precipitated with 2 volumes of ethanol, centrifuged and dissolved in 50 μl of 10 mM Tris-HCl, 1 mM EDTA, pH 7.6. This alkaline phosphatase treatment was performed with EcoRI and BamHI treated pBH20.
Effectively prevents self-association of DNA, but the conjugation still allows the formation of circular recombinant plasmids containing somatostatin DNA. E. FIG. coliR
The transformation of RI with linear plasmid DNA is so inefficient that the majority of this transformant will contain recombinant plasmids. Somatostatin DNA
50 μl (4 μg / ml) was added to BamHI at 22 ° C. in a total volume of 50 μl containing 20 mmol Tris-HCl, pH 7.6, 10 mmol MgCl _2, 10 mmol dithiothreate, 0.5 mmol ATP and 4 units T ₄ DNA ligase. ,
It was ligated with 25 μl of pBH20 DNA treated with EcoRI and alkaline phosphatase. After 10, 20 and 30 minutes, somatostatin DNA (40 ng) was added to the reaction mixture (gradual addition of somatostatin DNA is preferred to favor binding to the plasmid over self-binding). The conjugation reaction was continued for 1 hour and then the mixture was dialyzed against 10 mmol Tris-HCl pH 7.6. As a control experiment, pBH20 DNA treated with BamHI, EcoRI, alkaline phosphatase was ligated in the absence of somatostatin DNA under similar conditions. Both preparations were treated with E. coliR
It was used for transformation of RI. The transformation experiment was carried out in a P3 physical containment facility. [National Institutes of Health, National Institutes of Health
Health USA Recombinant DNA Research Guidelines
Guidelines) 1976]. Transformed strain Ap 20 μg /
ml and X-gal 40 μg / ml on minimal medium plates. Ten transformants were isolated which were all sensitive to Tc. For verification, these are pSOM
1, pSOM2 ..... was named pSOM10. No transformant was obtained in the control experiment. Four out of ten transformants contained a plasmid with both EcoRI and BamHI sites. The size of the small EcoRI, BamHI fragments of these recombinant plasmids was similar to the size of somatostatin DNA produced in vitro in all four cases. Proc. Nat. Acad. Sci. U.S. from Proc. Of the National Academy of Sciences of the United States of Maxam and Gilbert. .
A.) Nucleotide sequence analysis according to Vol. 74, page 560 (1977) revealed that the plasmid pSOM1 inserted the desired somatostatin DNA fragment.

DNA sequence analysis of the clone carrying the plasmid pSOM1 predicts that this clone should produce a peptide consisting of somatostatin. However, somatostatin radioimmunoreactivity was not detected in cell pellet extracts or extracts of culture supernatants, and the presence of somatostatin was also detected when 70% formic acid and cyanogen bromide were added directly to the growth medium. There wasn't. E. FIG. It has been observed that coliRRI extracts degrade exogenous somatostatin very quickly.
Therefore, it can be considered that the absence of somatostatin activity in the clone having the plasmid pSOM1 is due to the intracellular degradation by the endogenous protease. Therefore, we decided to use this plasmid pSOM1 to construct a plasmid containing a somatostatin and a code for a precursor protein of sufficient size to be expected to resist proteolysis.

E. Plasmid pSOM11 and pSO
Configuration of M11-3. In phase, we constructed a plasmid in which the somatostatin gene could be located at the C-terminus of the β-galactosidase gene. The existence of an EcoRI site near the C-terminus of this gene and the amino acid sequence of this protein are known [B. Polisky et al., Proceedings of the.
National Academy of Sciences of
The United States of America (Pro
c. Nat. Acad. Sci. USA) Volume 73, 3900
Page (1976), AV Fowler
er) et al., Id., 74, 1507 (1976), AI, Bukuhari et al., Nature New Biology.
Volume 243, 238 (1973) and Kay E.
Langley (KE Langley), Journal of Biological Chemistry (J. Biol. Chem.) 2nd
50, 2587 (1975)], the EcoRI BamHI somatostatin gene could be inserted at the EcoRI site while maintaining the proper reading frame. To construct such a plasmid, pSOM1 DNA (50 μm
g) was digested with the restriction enzymes EcoRI and PstI in a final volume of 100 μg. Using a preparative 5% polyacrylamide gel, a small fragment with the lac-regulatory element and a large Pst-EcoRI with the somatostatin gene.
The fragments were separated. The large band was cut from the gel, the gel was sliced into small pieces, and the DNA was eluted by extracting the DNA at 65 ° C. overnight. In a similar manner, plasmid pBR322 DNA (50 μg) was added to Pst
Digested with I and EcoRI restriction endonucleases, the two DNA fragments thus obtained were purified by electrophoresis on a preparative 5% polyacrylamide gel. Small PstI-EcoRI obtained from pBR322
Fragment (1 μg) was obtained from pSOM1 with large Pst
I-EcoRI DNA fragment (5 μg) and T ₄ DNA
One unit of ligase was used for ligation in a final volume of 50 μl for 12 hours at 12 ° C. This combined mixture was transformed into E. coli
Used for RRI transformation, the transformants were selected for ampicillin resistance on X-gal medium. As expected, almost all Ap ^r transformants (95%) gave white colonies (no lac-operator) on X-gal indicator plates. The plasmid pSOM11 thus obtained
Was used to construct plasmid pSOM11-3. pS
A mixture of 5 μg of OM11 DNA and 5 μg of λplac5 DNA was digested with EcoRI (10 units, 37 ° C. for 30 minutes). The digestion with this restriction endonuclease was terminated by phenol-chloroform extraction. Then this DN
A was precipitated with ethanol and T ₄ DNA ligase buffer
Resuspended (50 μl). T ₄ DNA ligase (1 unit)
Was added to this mixture and incubated at 12 ° C. for 12 hours. The combined mixture was dialyzed against 10 mM Tris-HCl, pH 7.6, E. It was used for transformation of the coli strain RRI. Transformed strains containing X-g containing ampicillin
We selected for Ap ^r on al plates and screened for constitutive β-galactosidase production. About 2% of the colonies were blue (pSOM11-1, 11-2, etc.). As a result of restriction enzyme analysis of the plasmid DNA obtained from these colonies, all of these plasmids had a new EcoRI fragment of about 4.4 megadalton, which was found to contain the lac-operon regulatory site and the β-galactosidase gene. It turned out to have the most. Since this EcoRI fragment can have two orientations (directions), the asymmetrical arrangement of the HindIII restriction sites indicates that any of these colonies undergo lac-transcription to the somatostatin gene. EcoR
Used to determine if it has the I fragment. Hin
As a result of double digestion of dIII-BamHI, plasmid pSO
M11-3, pSOM11-5, pSOM11-6 and
Only clones with pSOM11-7 were found to contain an EcoRI fragment with this orientation.

4. Radioimmunoassay for somatostatin activity Standard radioimmunoassay (RIA) for somatostatin [A. Arimura et al., Proceedings of the Society for Experimental]
Biology and Medicine (Proc. Soc. Ex
p. Biol. Med. ) Volume 148. Page 784 (1975)]
Was corrected by reducing the assay volume and using phosphate buffer. Tyr ¹¹ somatostatin was iodinated using the chloramine T method (Id.). For analysis of somatostatin, a sample, usually in 70% formic acid containing 5 mg / ml of cyanogen bromide, was placed in a conical polypropylene tube [0.7 ml, Sarstedt] on wet KOH. , Dried under reduced pressure. PBSA buffer (NaCl
75 mmol; sodium phosphate 75 mmol (pH 7.2);
Bovine serum albumin 1 mg / ml and sodium azide 0.2 mg / ml) 20 μl were added, followed by [ ¹²⁵ I] somatostatin “cocktail” 40 μl and rabbit anti-somatostatin immune serum S39 [Vale et al., Metabolism.
(Methabolism), Vol. 25, p. 1491 (1976)] was added to PBSA in a volume of 1,000 times and 20 μl thereof was added.
[ ¹²⁵ I] Somatostatin cocktail is 1 ml of PBSA buffer
250 μg of normal rabbit gamma globulin [Antibodies, Inc.],
Protease Inhibitor ["Tracylol"("Trasylo"
l "), Calbiochem] 1500 units and [
^It contained approximately 100,000 counts of ¹²⁵ I] Tyr ¹¹ -somatostatin. Goat anti-rabbit gamma globulin in PBSA buffer after at least 16 hours at room temperature
(Antibodies Incorporated P = 0.0
3) 0.333 ml was added to the sample tube. The mixture was incubated at 37 ° C for 2 hours, cooled to 5 ° C,
It was then centrifuged at 10,000 xg for 5 minutes. The supernatant was removed and the pellets were counted with a γ counter. With the amount of antiserum used, 20% of the counts precipitated without unlabeled competing somatostatin. A large amount of somatostatin (200n
The background in g) was usually 3%. Half maximal competition was obtained with 10 pg somatostatin. E. FIG. Initial experiments with extracts of the coli strain RRI (recipient strain) revealed that 10 pg or less of somatostatin can be easily detected in the presence of 16 μg or more of cyanogen bromide treated bacterial protein. Over 2 μg of protein from formic acid-treated bacterial extracts interfered somewhat with increasing background, whereas cyanogen bromide cleavage greatly reduced this interference. Reassembly experiments have shown that somatostatin is stable in cyanogen bromide treated extracts.

A. Competing strain E. coli with bacterial extract ColiRRI (pSOM11-5) and E. Col
iRRI (pSOM11-4) was grown in Luria broth at 37 ° C. to 5 × 10 ⁸ cells / ml. Then 1 mmol of IPTG was added and growth was continued for 2 hours. 1 ml, which is a part of it (Aliquot), is used in Eppendorf.
Centrifuge for several seconds in a centrifuge and pellet the pellet with 500 μl of 70% formic acid containing 5 mg / ml of cyanogen bromide.
Was suspended. After about 24 hours at room temperature, this aliquot was diluted 10-fold with water, and somatostatin was measured three times in the volume shown in FIG. In FIG. 6, [B / Bo] is the ratio of [ ¹²⁵ I] somatostatin bound in the presence of sample to [ ¹²⁵ I] somatostatin bound in the absence of competing somatostatin. Symbol 1 is E. coli RRI
(PSOM11-4), 2 is E. coli RRI (pSOM1
1-5) is shown. Each point is the average of 3 tubes. The protein content of the undiluted sample is
E. FIG. 2.2 mg / ml for ColiRRI (pSOM11-5),
E. FIG. It was 1.5 mg / ml by ColiRRI (pSOM11-4).

B. PSOM11 claw for somatostatin
First screening 11 clones (pSOM11-2, pSOM11-3, etc.)
The cyanogen bromide treated extract of was prepared as described for the case of FIG. 30 μl of each extract was collected and subjected to radioimmunoassay three times. FIG. 7 shows the result. In the figure, the range of analytical values is shown. Somatostatin pyrogram values were read from a standard curve obtained as part of the same experiment.

The results of the radioimmunoassay described so far can be summarized as follows. Unlike the experimental results with pSOM1, four clones (pSOM11-3, 11
-5, 11-6 and 11-7) were found to have readily detectable somatostatin radioactivity (Fig. 6).
And Figure 7). As a result of restriction fragment analysis, pSOM1
1-3, pSOM11-5, pSOM11-6 and pS
OM11-7 was found to have the desired lac-operon orientation, while pSOM11-2 and 11-4 had the opposite orientation. Thus, there is a perfect correlation between the correct lac-operon orientation and the production of somatostatin radioimmunoreactivity.

C. IP on positive and negative clones
Effects of TG induction and CNBr cleavage In the design of this somatostatin plasmid ,
Somatostatin was expected to be synthesized under the control of the lac-operon. The lac-repressor gene was not included in this plasmid and
(E. ColiRRI) contains the wild-type chromosomal lac-repressor gene producing only 10-20 repressor molecules per cell. The copy number of this plasmid (and thus the number of lac-operators) is approximately 2 per cell.
Since it is 0 to 30, complete suppression is impossible. As shown in Table 3 below, E. coli RRI (pSOM11-3)
The specific activity of somatostatin was increased by IPTG, the inducer of the lac-operon. As expected, the level of induction was low, 2.4-7 fold. Experiment 7
In Table 3, the α activity, which is an indicator of the first 92 amino acids of β-galactosidase, was also doubled. In some experiments, somatostatin radioimmunoreactivity was undetectable before cleavage of whole cell proteins with cyanogen bromide. The antiserum S39 used in the radioimmunoassay required free N-terminal alanine, so no activity was expected before cleavage with cyanogen bromide.

[0069]

Table 3 Somatostatin radioimmunospecific activity Abbreviations: Luria Broth, LB; Isopropylthiogalactoside, IPTG; Cyanogen bromide, CNBr; Somatostatin, SS. Protein is Bradhord (Br
Adford), Analytical Biochemistry (Anal. Bi
ochem.) 72, 248 (1976). Experiment IPTG CNBr pgSS / μg No. Strain Medium 1 mM 5 mg / ml Protein 1 11-2 LB + + <0.1 11-3 LB + + 12 11-4 LB + + <0.4 11-5 LB + + 15 2 11-3 LB + + 12 11-3 LB +-<0.1 3 11-3 LB + + 61 11-3 LB- + 8 11-3 LB +-<0.1 4 11-3 LB + + 71 11-3 VB + Glycerol * + +62 5 11-3 LB + Glycerol + + 250 6 11-3 LB + + 320 11-2 LB + + <0.1 7 11-3 LB + + 24 11-3 LB- + 10 * Vogel-Bonnel (Vogel- Bonner) Minimal medium + glycerol.

D. Gel filtration of cyanogen bromide treated extracts (pSOM 11-3, 11-5, 11-6 and 11-7) formic acid and cyanogen bromide treated extracts were pooled (total volume 250 μl), dried and % Acetic acid 0.1 ml
Was resuspended. [ ³ H] leucine was added and the sample was packed with Sephadex G-50 in 50% acetic acid to 0.7.
It was applied to a × 47 cm column. A 50 μl portion of the column fraction was taken and analyzed for somatostatin. Pooled negative clone extracts (11-2, 11-4 and 11-11) were treated in a similar manner. The results are shown in Fig. 8. The symbol 3 indicates a pooled positive clone, 4 indicates ³ H-Leu used as a control, and 5 indicates a pooled negative clone. Somatostatin known in the same column [Beckman Corp.]
Elute as indicated by arrow (6). In this system,
Somatostatin is well separated from the excluded large peptide and the fully encapsulated small molecule. Only extracts of clones positive for somatostatin show radioimmunoactivity in the column fractions, the active part eluting at the same position as chemically synthesized somatostatin.

Summary of Activity Information The information for carrying out the synthesis of polypeptides containing the amino acid sequence of somatostatin is summarized as follows.
(1) Somatostatin radioimmunoactivity contains the proven correct sequence somatostatin gene,
Present in E. coli cells containing the plasmid pSOM11-3 with the lac-EcoRI DNA fragment.
Has the same somatostatin gene, but with lac-EcoRI
Cells with plasmid pSOM11-2 associated with it, which has the opposite fragment orientation, show no detectable somatostatin activity. (2) Somatostatin radioimmunoreactivity is not observed until the cell extract is treated with cyanogen bromide, as expected in the design proposal. (3)
Somatostatin activity is controlled by the lac-operon, as evidenced by its induction by the lac-operon inducer IPTG. (4)
The somatostatin active part is co-chromatographed together with known somatostatin on Sephadex G-50. (5) D of cloned somatostatin gene
The NA sequence is correct. If the translation is out of phase, a peptide that differs from somatostatin at any position will be produced. The detection of radioimmunoreactivity indicates production of a polypeptide closely related to somatostatin, thus indicating that translation should be in phase. Since translation occurs out of phase, the genetic code directs the production of peptides with somatostatin in the correct sequence order. (6) Finally,
Said sample of E. coli RRI (pSOM11-3) extract inhibits growth hormone secretion from rat pituitary cells, while E. coli RRI (pSOM11- The sample of 2) has no effect on the secretion of growth hormone.

Stability, yield and purification of somatostatin EcoRIlac-operon fragment (pSOM11-
2, pSOM11-3, etc.) are divergent with respect to the phenotype of the plasmid. For example, after about 15 generations, about half of the E. coli RRI (pSOM11-3) cultures constituted β-galactosidase (ie had the lac-operator) and about half of these were ampicillin resistant. Somatostatin positive (pSOM11-3) and negative (pSOM11-2) strains are unstable. The reason why this growth is disadvantageous is therefore great, but
It is likely due to incomplete and inactive galactosidase overproduction. The somatostatin yield is probably lac-
It appears to be the result of selecting cells from cultures with region-deficient plasmids, but varied between 0.001 and 0.03% relative to total cellular protein (Table 3). The highest yields of somatostatin are obtained when growing from a single ampicillin-resistant strain, ie the constituent colonies.
Even in these cases, 30% of the cells at harvest were lacking the lac-region. The method of storage in the frozen state (freeze-dried) and growing and harvesting from a single such colony is therefore indicated as the system described thus far. The yield may be increased by choosing a bacterial strain that overproduces the lac-repressor such that expression of the precursor protein is essentially completely suppressed until before induction and harvest. Alternatively, tryptophan or other operator-promoter system, which is usually completely repressed, may be used, as described above.

For example, Eaton Pres
The β-galactosidase-somatostatin precursor protein is insoluble in the crude extract obtained by cell disruption in s) and is therefore found in the pellet of the first low speed centrifugation. This activator is 70% formic acid, 6
It can be dissolved in molar guanidinium hydrochloride or 2% sodium dodecyl sulfate. However, it is most preferred to extract the crude extract from Eaton Press with 8 moles of urea and cleave the residue with cyanogen bromide. In the first experiment, somatostatin activity obtained from E. coli strain RRI (pSOM11-3) was obtained by extracting the cleavage product with alcohol,
Chromatography on Sephadex G-50 with 50% acetic acid increased approximately 100-fold. The product was again chromatographed on Sephadex G-50, followed by high pressure liquid chromatography,
Substantially pure somatostatin is obtained.

II Human Insulin The techniques described above were then applied to the production of human insulin. That is, a gene for insulin B chain (104 base pairs) and a gene for insulin A chain (77 base pairs) were designed from the amino acid sequence of human polypeptide. Each chain has EcoRI and BamHI
It was designed to have single-stranded sticky ends for the restriction endonucleases and to be inserted separately into the pBR322 plasmid. Using trinucleotides as building blocks, the synthetic fragment deca or pentadecanucleotide was synthesized by the block phosphate triester method and finally purified by high performance liquid chromatography (HPLC). Then, this human insulin A and B chain synthesis gene was cloned into plasmid p.
Separately cloned in BR322. The cloned synthetic gene was fused with E. coli β-galactosidase as described above, and was effectively transcribed and translated to obtain a stable precursor protein. The insulin peptide was cleaved from the β-galactosidase precursor, detected by radioimmunoassay and purified. Insulin radioimmunoreactivity was then produced by mixing with E. coli product.

1. Human insulin gene design and
Figure 9 shows the gene assembled for synthetic human insulin.
Shown in The gene for human insulin, the B chain and the A chain were designed from the amino acid sequence of the human polypeptide. Each gene is plasmid pBR322
There is a single-stranded sticky end for the EcoRI and BamHI restriction endonucleases at the 5'end of each gene for correct insertion into. Prior to constructing the entire B chain gene, a HindIII endonuclease recognition site was inserted in the center of the B chain gene so that the amino acid sequence Glu-ala would allow amplification and confirmation of each half gene. The B chain and A chain genes are
It was designed to be assembled from 29 different oligodeoxyribonucleotides ranging from decamers to pentadecamers. Each arrow represents a fragment synthesized by the modified phosphate triester method. H ₁
To H ₈ and B ₁ to B ₁₀ are for the B chain gene,
A ₁ to A ₁₂ are for A chain genes.

2. Chemistry of oligodexoxyribonucleotides
Synthesis The methods and raw materials for oligodexoxyribonucleotide synthesis were basically reported by Itakura et al., Except for the changes described below (Itakura, K. et al.
(1975) J. Biol. Chem. 250 4592 and Itakura, K. et al (1975) J. Amer. Chem. So
c. 97, 7327).

A) a fully protected mononucleotide,
5'-O-dimethoxytrityl-3'-p-chlorophenyl-β-cyanoethylphosphate is a monofunctional phosphorylating agent, p-chlorophenyl-β-cyanoethylphosphorochloridate, in acetonitrile in the presence of 1-methylimidazole. (1.5 molar equivalents) was used to synthesize from nucleotide derivatives (Vam Boom, JH et al (197).
5) Tetrahedron 31, 2953). The product was purified by preparative liquid chromatography (Prep 500LC, Waters
Large scale (100-300 g) isolation by Associates).

B) Solvent extraction method (Hirose, T. et al (19)
78) Tetrahedron Letters, 2449), 3
Two bifunctional trimers (see Table 4) were synthesized on a 5-10 mmol scale, 4 dimers as 3'-end blocks, 3 tetramers and 13 trimers on a 1 mmol scale. The homogeneity of the fully protected trimer is found in two methanol / chloroform solvent systems, namely solvent a at 5% V / V and solvent b at 10%.
Confirmed by thin layer chromatography on silica gel using% V / V (see Table 4). Starting from this group of compounds, 29 oligodeoxyribonucleotides with a fixed sequence were synthesized, namely 18 for the B chain and 11 for the A chain gene.

The base units used to construct the polynucleotide were two types of trimer blocks, namely the bifunctional trimer block of Table 4 and the corresponding 3'-terminal trimer with the 3'-hydroxyl group protected by an anisoyl group. there were. This bifunctional trimer was converted to the corresponding 3'by pyridine-triethylamine-water (3: 1: 1 V / V) mixture.
-Hydrolyzed to the phosphoric acid diester component and also to the corresponding 5'-hydroxyl component with 2% benzenesulfonic acid. The 3'-end block described above was treated with 2% benzenesulfonic acid to give the corresponding 5'-hydroxyl form. The condensation reaction of an excess of this 3'-phosphoric acid diester trimer (1.5 molar equivalents) with the 5'-hydroxyl component (1 molar equivalents) gives 2,4,
6-triisopropylbenzenesulfonyl tetrazolide
(TPSTe, 3-4 equivalents) and was almost completely completed in 3 hours. The reaction mixture was passed through a short silica gel column set on a semi-molten glass filter to remove excess 3'-phosphodiester block reactant. From this column, first elute and wash with chloroform to elute the by-products and condensing agent, then CH.
Elution with Cl ₃ : MeOH (95: 5 V / V) eluted most of the fully protected oligomer. Under this condition, the 3'-phosphodiester block reactant passed through the column remained on the column. Similarly, block coupling was repeated until the desired length was obtained.

[0080]

[Table 4] * Fully protected trideoxynucleotide;
5-0-Dimethoxytrityl-3′-p-chlorophenyl-β-cyanoethylphosphoric acid ** Yield is the total yield calculated from 5′-hydroxyl monomer. *** Based on HPLC analysis.

In oligonucleotide synthesis, a) analysis of the respective trimer and tetramer blocks, b) intermediate fragments (hexamers, nonamers and decamers)
C) analysis of the last condensation reaction, d) purification of the final product,
High performance liquid chromatography (HPLC) was extensively used for This HPLC is based on Spectra-Physics
Performed using a 3500B Liquid Chromatograph.
After removal of all protecting groups with concentrated NH ₄ OH at 50 ° C. (6 h) and 80% AcOH at room temperature (15 min), in solvent A (0.01 M KH ₂ PO ₄ , pH 4.5). ,
Solvent B (0.05 MKH ₂ PO ₄ -1.0 M KCl, pH 4.
5) using the linear inclination method according to Perm Phase AAX [Van Boom, J. et al (1977) J. Chr
Compounds were analyzed on omatography 131 169] column (1 m × 2 mm). Gradients were started by starting with buffer A and adding 3% buffer B every minute. Elution is 6
It was carried out at 0 ° C. with a flow rate of 2 ml per minute. Purification of the 29 last oligonucleotides was also performed on Permaphase AAX under the same conditions as above. The target peak was collected, desalted by dialysis, and freeze-dried.
(Γ- ³² P) AT using T ₄ polynucleotide kinase
After labeling the 5'-end with P, the homogeneity of each oligonucleotide was checked by electrophoresis on a 20% polyacrylamide gel.

[0082] 3. Assembly of B chain gene and A chain gene
And cloning The gene for the B chain of insulin has an EcoR at the left end.
I restriction site, HindIII site in the center, and Bam at the right end
It was designed to have a HI site. This is done by cloning the halves of both, namely the EcoRI-HindIII half on the left (BH) and the HindIII-BamHI half on the right (BB), separately into a suitable cloning vehicle pBR322 and confirming their sequence. After that, it was carried out so that they could be combined into a complete B gene (Fig. 1).
0). This BB half is designated as B1 through B1 in FIG.
It was assembled by ligating 10 oligodeoxyribonucleotides, numbered 0, prepared by phosphotriester chemical synthesis. B1 and B10 were not phosphorylated so that these fragments did not undergo unwanted polymerization due to sticky ends (HindIII and BamHI). After purification by preparative acrylamide gel electrophoresis and elution of the largest DNA band, this BB fragment was inserted into HindIII and BamHI cleaved plasmid pBR322. Approximately 50% of ampicillin resistant colonies derived from this DNA were sensitive to tetracycline, indicating the insertion of the non-plasmid HindIII-BamHI fragment. 4 of these colonies (pBB
101-pBB104) small HindIII-BamHI
The fragments were sequenced and found to be correct as designed.

A BH fragment was prepared in the same manner and E
It was inserted into pBR322 that had been cleaved with coRI and HindIII restriction endonucleases. Three tetracycline-sensitive transformants from ampicillin-resistant plasmid
(pBH1 to pBH3) were analyzed. This little EcoRI-
The HindIII fragment was found to have the desired nucleotide sequence.

The A chain gene was assembled from three parts.
Four on the left, four in the middle and four on the right (see FIG. 9) were ligated separately, then mixed and ligated (oligonucleotides A1 and A12 were not phosphorylated). The assembled A chain gene was phosphorylated and purified by gel electrophoresis, pBR322, Ec
It was cloned into the oRI-BamHI site. The EcoRI-BamHI fragment from two ampicillin resistant, tetracycline sensitive clones (pA10 and pA11) contained the desired A gene sequence.

[0085] 4. Expression of A and B insulin genes
Assembly of plasmids for lac-insulin B plasmid (pIB1)
It shows the assembly of. Plus mid pBH1
And pBB101 were digested with EcoRI and HindIII endonucleases. This small BH fragment of pBH1 and the large fragment of pBB101 (BB
The fragment and most of pBR322) was purified by gel electrophoresis, mixed and EcoRI.
Ligation was performed in the presence of λplac5 cleaved at This λplac
The 5 megadalton EcoRI fragment contains the lac-regulatory region and most of the β-galactosidase structural gene. The placement of this restriction site ensures the correct binding of BH to BB. There are two insertion directions of this lac-EcoRI fragment. Therefore, only half of the clones obtained after transformation should have the desired orientation. 10 showing ampicillin resistance and β-
The orientation of clones constitutively producing galactosidase was examined by restriction analysis. 5 of these colonies
Each had the complete B gene sequence and the correct open reading frame from the β-galactosidase gene to the B chain gene. One of them, pIB1, was chosen for the next experiment.

By the same experiment, 4. from λplac5.
The 4 megadalton lac-fragment was introduced into the EcoRI site of pA11 plasmid to give pIA1. pIA1
Is the same as pIB1 except that the A gene fragment replaces the B gene fragment. As a result of DNA sequence analysis, it was found that the correct A and B chain gene sequences were retained in pIA1 and pIB1, respectively.

5. Both strains containing the insulin gene correctly attached to the expressed β-galactosidase produce large amounts of β-galactosidase large protein. Approximately 20% of total cellular protein is this β-galactosidase-insulin A
Or it was a B-chain hybrid. The hybrid proteins are insoluble and are present in the first slow pellet, where they make up about 50% of the protein.

To detect the expression of the insulin A and B chains, we use a radioimmunoassay (RIA) based on restoring complete insulin from this separate chain.
It was used. Insulin restoration method of Katsoyannis et al. (Katsoy) using 27 μl assay volume.
annis et al (1967) Biochemistry 6 , 2642-
2655) is a very suitable assay method. After mixing the insulin chains and reconstituting the S-Sulfonated derivative, the insulin activity is easily detected. The separate S-sulfonated chains of insulin do not react significantly with the anti-insulin antibody used after reduction and oxidation.

To use this refolding assay, a portion of the β-galactosidase-A or B chain hybrid protein was purified and cleaved with cyanogen bromide to form the S-sulfonated derivative.

Evidence that correct expression was obtained from a chemically synthesized gene for human insulin can be summarized as follows. a) Radioimmunoreactivity was detected in both chains. b) The DNA sequence and plasmid construction obtained after cloning was directly confirmed to be correct as designed. Translation must be in phase as radioimmunoreactivity is obtained. Therefore, the genetic code dictates that peptides with the sequence of human insulin be produced. c) The E. coli product after cleavage with cyanogen bromide behaves as an insulin chain in three different chromatographic systems (gel filtration, ion exchange and reverse phase HPLC) based on different principles. It was d) E. coli produced A chain was purified by HPLC on a small scale. And it has the correct amino acid composition.

[Brief description of the drawings]

FIG. 1 is a flow chart for biological production of somatostatin.

FIG. 2 is a diagram showing a schematic structure of a structural gene.

FIG. 3 is a flow chart for nucleotide trimer preparation.

FIG. 4 is a flow chart for preparing recombinant plasmid pSOM11-3 from parental plasmid pBR322.

[Fig. 5] Fig. 5 is a diagram showing a nucleotide sequence that is a key part of two plasmids.

FIG. 6 is a graph showing somatostatin activity of products expressed by recombinant plasmids.

FIG. 7 is a graph showing somatostatin activity of products expressed by recombinant plasmids.

FIG. 8 is a graph showing somatostatin activity of products expressed by recombinant plasmids.

FIG. 9 is a diagram showing a schematic structure of a synthetic gene consisting of codons for the amino acid sequences of human insulin A and B chains.

FIG. 10 is a flow chart for constructing a recombinant plasmid expressing the B chain of human insulin. 1 ... pSOM11-4, 2 ... pSOM11-5, 3 ...
Pooled positive clones, 4 ... ³ H-Leu, 5 ... Pooled negative clones, 6 ... Arrows indicating positions where known somatostatin elutes.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:19) (56) References PROC. NATL. ACAD. SCI. USA, VOL. 73, NO. 11 (1976) P. 3900-3904 SCIENCE, VOL. 196 (17 JUNE 1977) P. 1313-1319

Claims (1)

(57) [Claims] A method for producing a polypeptide containing a predetermined functional mammalian polypeptide or an intermediate polypeptide thereof in a microbial cell culture, comprising the steps of: (i) Expressing the polypeptide by a microorganism transformed with a replicable cloning vehicle containing DNA under the control of a homologous expression control region, then (ii) recovering the polypeptide from the cell culture, A method consisting of things. 2. The method according to claim 1, wherein the DNA is cDNA. 3. The method according to claim 1, wherein the DNA is chemically synthesized in vitro. 4. The method according to claim 3, wherein the microorganism is a bacterium, and the DNA contains a codon preferable for the bacterium. 5. The method according to claim 1, wherein the expression control region is inducible, and the expression of the polypeptide is suppressed by the expression control region until the expression is induced. 6. The polypeptide comprises a serum protein.
The method described in. 7. The method of claim 1, wherein the polypeptide is a hormonally active mammalian hormone. 8. The method according to claim 1, wherein the expression control region comprises a part of an operon. 9. The method of claim 1 wherein the polypeptide comprises a mammalian polypeptide consisting essentially of the human insulin A or B chain. 10. The method of claim 1, wherein the polypeptide is recovered from the microorganism in an insoluble form. 11. The method of claim 1, wherein the polypeptide comprises a mammalian polypeptide and a selective cleavage site adjacent to the mammalian polypeptide. 12. 7. The method according to claim 6, wherein the serum protein is expressed without additional proteins preceding or following it, and is capable of resisting intracellular degradation by the endogenous proteolytic enzyme of the microorganism. 13. 2. A start codon before the DNA and one or more stop codons immediately after the DNA.
The method described in. 14． 14. The method of claim 13, wherein the polypeptide is produced as such. 15. The method according to claim 1, wherein the DNA is chemically synthesized in vitro. 16. The polypeptide is a mammalian polypeptide without preceding and / or following additional proteins, which is capable of resisting intracellular degradation by the endogenous proteolytic enzyme of the microorganism, the expression control region 16. The method of claim 15, wherein the comprises a portion of the operon. 17． The method according to claim 16, wherein the expression control region is inducible, and the expression of the polypeptide is controlled by the expression control region before the induction of expression. 18. 18. The method of claim 17, wherein the microorganism is a bacterium and the DNA consists of codons preferred for the bacterium. 19. 17. The method of claim 16, wherein the polypeptide comprises serum protein. 20. 17. The method of claim 16, wherein the polypeptide comprises a hormonally active mammalian hormone. 21. 16. The method of claim 15, wherein the polypeptide comprises a mammalian polypeptide and a selective cleavage site adjacent to the polypeptide. 22. 22. The method of claim 21, wherein the mammalian polypeptide is the human insulin A or B chain. 23. 19. The method of claim 18, wherein the polypeptide is recovered from the microorganism in an insoluble form. 24. The method of claim 1, wherein the polypeptide is recovered from the cell culture in insoluble form. 25. 25. The method of claim 24, wherein the DNA comprises cDNA. 26. 25. The method of claim 24, wherein the DNA is chemically synthesized in vitro. 27. 27. The method of claim 26, wherein the DNA comprises bacterial preferred codons. 28. 25. The method of claim 24, wherein the polypeptide comprises a mammalian polypeptide and a selective cleavage site adjacent to the polypeptide. 29. 25. The polypeptide according to claim 24, wherein the polypeptide is a mammalian polypeptide without preceding and / or following additional proteins, which is capable of resisting intracellular degradation by an endogenous proteolytic enzyme of the microorganism. The method described. 30. The polypeptide is substantially A of human insulin.
29. The method of claim 28, comprising a mammalian polypeptide consisting of a chain or a B chain. 31. 29. The method of claim 28, wherein the polypeptide comprises a serum protein. 32. 29. The method of claim 28, wherein the polypeptide comprises a mammalian hormone having hormonal activity. 33. 25. The method of claim 24, wherein the expression control sequence comprises a portion of the operon. 34. The DNA encodes a mammalian polypeptide, without additional proteins preceding and / or following, which is resistant to intracellular degradation by the endogenous proteolytic enzyme of the microorganism. The method according to Item 1. 35. 35. The method of claim 34, wherein the polypeptide is a mammalian polypeptide and is recovered from the microorganism in an insoluble form. 36. 36. The method of claim 35, wherein the DNA comprises cDNA. 37. 36. The method of claim 35, wherein the DNA is chemically synthesized in vitro. 38. 38. The method of claim 37, wherein the microorganism is a bacterium and the DNA contains codons preferred for the bacterium. 39. 36. The method of claim 35, wherein the expression control sequence is inducible and the expression of the polypeptide is suppressed under the expression control region prior to the induction of expression. 40. 39. The method of claim 38, wherein the polypeptide comprises serum protein. 41. 39. The method of claim 38, wherein the polypeptide is a mammalian hormone having hormonal activity. 42. 42. The method of claim 41, wherein the expression control region comprises a portion of the operon. 43. 31. The method of any of claims 22-30, wherein the replicable cloning vehicle is pIA1. 44. 31. The method of any of claims 22-30, wherein the replicable cloning vehicle is pIB1. 45. The DNA (a) encodes a polypeptide capable of resisting intracellular degradation by an endogenous proteolytic enzyme of the microorganism without (a) additional proteins preceding and / or following, (b) 2.) The method according to claim 1, wherein the polypeptide is chemically synthesized in vitro and the polypeptide is recovered from the cell culture in an insoluble form. 46. A method of producing a polypeptide in a microbial cell culture comprising a predetermined functional mammalian polypeptide, comprising: (i) a polypeptide without additional proteins preceding and / or following. And chemically synthesizing in vitro a DNA encoding a polypeptide capable of resisting intracellular degradation by an endogenous protease of the microorganism, and (ii) making the DNA homologous to the microorganism. It is ligated to a replication-competent cloning vehicle so as to be under the control of an expression control sequence, (iii) the polypeptide is expressed in a microorganism transformed with the cloning vehicle, and (iv) the polypeptide is cultured in a cell. A method that consists of recovering from.